CN114675028B - Preparation method and application of fenitrothion nano-antibody-colloidal gold marker - Google Patents
Preparation method and application of fenitrothion nano-antibody-colloidal gold marker Download PDFInfo
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- CN114675028B CN114675028B CN202210357015.8A CN202210357015A CN114675028B CN 114675028 B CN114675028 B CN 114675028B CN 202210357015 A CN202210357015 A CN 202210357015A CN 114675028 B CN114675028 B CN 114675028B
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- colloidal gold
- fenitrothion
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- 239000003550 marker Substances 0.000 title claims abstract description 49
- ZNOLGFHPUIJIMJ-UHFFFAOYSA-N fenitrothion Chemical compound COP(=S)(OC)OC1=CC=C([N+]([O-])=O)C(C)=C1 ZNOLGFHPUIJIMJ-UHFFFAOYSA-N 0.000 title claims description 133
- 238000002360 preparation method Methods 0.000 title abstract description 28
- 238000001514 detection method Methods 0.000 claims abstract description 65
- 238000012360 testing method Methods 0.000 claims abstract description 61
- 239000000575 pesticide Substances 0.000 claims abstract description 54
- 238000000034 method Methods 0.000 claims abstract description 17
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 69
- 239000000243 solution Substances 0.000 claims description 61
- 238000006243 chemical reaction Methods 0.000 claims description 29
- 238000010521 absorption reaction Methods 0.000 claims description 21
- 239000011248 coating agent Substances 0.000 claims description 18
- 238000000576 coating method Methods 0.000 claims description 18
- 239000012528 membrane Substances 0.000 claims description 14
- 238000003908 quality control method Methods 0.000 claims description 14
- 238000007789 sealing Methods 0.000 claims description 14
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 12
- 229930006000 Sucrose Natural products 0.000 claims description 11
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 11
- 239000005720 sucrose Substances 0.000 claims description 11
- 229940078795 lactoferrin Drugs 0.000 claims description 10
- 239000001509 sodium citrate Substances 0.000 claims description 10
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 10
- 229940038773 trisodium citrate Drugs 0.000 claims description 10
- IRUJZVNXZWPBMU-UHFFFAOYSA-N cartap Chemical compound NC(=O)SCC(N(C)C)CSC(N)=O IRUJZVNXZWPBMU-UHFFFAOYSA-N 0.000 claims description 9
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- GFNANZIMVAIWHM-OBYCQNJPSA-N triamcinolone Chemical compound O=C1C=C[C@]2(C)[C@@]3(F)[C@@H](O)C[C@](C)([C@@]([C@H](O)C4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 GFNANZIMVAIWHM-OBYCQNJPSA-N 0.000 claims 1
- 229960005294 triamcinolone Drugs 0.000 claims 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 15
- 238000012216 screening Methods 0.000 abstract description 4
- 238000012544 monitoring process Methods 0.000 abstract description 3
- 238000003860 storage Methods 0.000 abstract description 3
- 230000035484 reaction time Effects 0.000 abstract description 2
- 239000005946 Cypermethrin Substances 0.000 abstract 6
- KAATUXNTWXVJKI-UHFFFAOYSA-N cypermethrin Chemical compound CC1(C)C(C=C(Cl)Cl)C1C(=O)OC(C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 KAATUXNTWXVJKI-UHFFFAOYSA-N 0.000 abstract 6
- 229960005424 cypermethrin Drugs 0.000 abstract 6
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- 239000000020 Nitrocellulose Substances 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 3
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- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 240000008067 Cucumis sativus Species 0.000 description 2
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 239000005916 Methomyl Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
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- DUEPRVBVGDRKAG-UHFFFAOYSA-N carbofuran Chemical compound CNC(=O)OC1=CC=CC2=C1OC(C)(C)C2 DUEPRVBVGDRKAG-UHFFFAOYSA-N 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
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- 238000010586 diagram Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- UHXUZOCRWCRNSJ-QPJJXVBHSA-N methomyl Chemical compound CNC(=O)O\N=C(/C)SC UHXUZOCRWCRNSJ-QPJJXVBHSA-N 0.000 description 2
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- 239000002105 nanoparticle Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- QYMMJNLHFKGANY-UHFFFAOYSA-N profenofos Chemical compound CCCSP(=O)(OCC)OC1=CC=C(Br)C=C1Cl QYMMJNLHFKGANY-UHFFFAOYSA-N 0.000 description 2
- URKOMYMAXPYINW-UHFFFAOYSA-N quetiapine Chemical compound C1CN(CCOCCO)CCN1C1=NC2=CC=CC=C2SC2=CC=CC=C12 URKOMYMAXPYINW-UHFFFAOYSA-N 0.000 description 2
- 229960004431 quetiapine Drugs 0.000 description 2
- 238000013102 re-test Methods 0.000 description 2
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- 239000011780 sodium chloride Substances 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
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- AMFGTOFWMRQMEM-UHFFFAOYSA-N triazophos Chemical compound N1=C(OP(=S)(OCC)OCC)N=CN1C1=CC=CC=C1 AMFGTOFWMRQMEM-UHFFFAOYSA-N 0.000 description 2
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- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
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Abstract
本发明公开了一种杀螟硫磷纳米抗体‑胶体金标记物的制备方法和应用。本发明提供了杀螟硫磷纳米抗体‑胶体金标记物的制备方法，制备得到的杀螟硫磷纳米抗体‑胶体金标记物的灵敏度高、稳定性强。本发明提供的检测杀螟硫磷农药的纳米抗体‑胶体金试纸条，具有灵敏度高、特异性强、成本低、操作简单、检测时间短、适用于市场的快速检测、易于储存、保质期长的优点。采用本发制备得到的纳米抗体‑胶体金试纸条在对杀螟硫磷农药的残留检测中，灵敏度达到0.013～0.309mg/kg，反应时间为5～8min，检测杀螟硫磷农药残留的方法简便、快速、直观、准确、适用范围广、成本低、易推广使用，适合市场上大量样本的筛查和现场监控。
The present invention discloses a preparation method and application of a cypermethrin nano-antibody-colloidal gold marker. The present invention provides a preparation method of a cypermethrin nano-antibody-colloidal gold marker, and the prepared cypermethrin nano-antibody-colloidal gold marker has high sensitivity and strong stability. The nano-antibody-colloidal gold test strip for detecting cypermethrin pesticide provided by the present invention has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, rapid detection suitable for the market, easy storage, and long shelf life. The nano-antibody-colloidal gold test strip prepared by the present invention has a sensitivity of 0.013-0.309 mg/kg in the residual detection of cypermethrin pesticide, and the reaction time is 5-8 min. The method for detecting the residue of cypermethrin pesticide is simple, rapid, intuitive, accurate, widely applicable, low cost, easy to promote and use, and is suitable for the screening and on-site monitoring of a large number of samples on the market.
Description
Technical Field
The invention belongs to the technical field of immunoassay detection of pesticide residues. More particularly relates to a preparation method and application of a fenitrothion nano antibody-colloidal gold marker.
Background
Fenitrothion (Fenitrothion) is also called fenitrothion, is an organophosphorus broad-spectrum pesticide, has very wide application range, and can be generally used for preventing and controlling pests of rice, wheat, oil crops, fruits and vegetables and trees. The fenitrothion belongs to a moderately toxic pesticide, the residual accumulation of the fenitrothion on crops can be caused by unreasonable application of the fenitrothion, and excessive intake of the fenitrothion has certain toxic effects on people and livestock, namely, single bond accumulation in a nervous system is caused, nausea, vomiting, abdominal pain, diarrhea, coma and other symptoms are caused, and the acute poisoning can endanger life. In recent years, the standard exceeding of fenitrothion in the screening of food samples in China is most serious, and the detection rate is higher than that of similar organophosphorus pesticides. Therefore, the development of a more economic, efficient and sensitive rapid detection technology for fenitrothion is of great significance in monitoring the fenitrothion.
The current standard detection method of fenitrothion is mainly an instrument analysis method and a epidemic analysis method. The instrument analysis method has accurate and sensitive detection results, can realize simultaneous detection of various objects to be detected, but needs complex and complicated sample pretreatment to reduce interference of a sample matrix on the analysis results, and needs expensive reagents and equipment and skilled technicians, which all cause the instrument analysis method to be unsuitable for large-scale instant detection in the market. The immunochromatography is to fix a specific antibody to a certain zone of a nitrocellulose membrane, after one end of the dried nitrocellulose is immersed in a sample (liquid), the sample moves forward along the membrane due to capillary action, and when the sample moves to a zone where the antibody is fixed, the corresponding antigen in the sample is specifically combined with the antibody, and if the zone is stained with immune colloidal gold or immune enzyme, the zone can display a certain color, thereby realizing specific immunodiagnosis. The immunoassay method has the advantages of strong specificity, high sensitivity, low detection cost, short detection time, low requirement on personnel and the like, and as CN106290830A in the prior art discloses an immunochromatographic test paper for rapidly detecting fenitrothion based on up-conversion fluorescent nanoparticles and a preparation method thereof, the antibody sample marked by the up-conversion fluorescent nanoparticles is used for detecting fenitrothion pesticides, and monoclonal antibodies or polyclonal antibodies are combined with colloidal gold for immunochromatographic detection, but the method is used for detecting residues of various organophosphorus pesticides. However, the antibodies used in the prior art of colloidal gold immunochromatography are almost all monoclonal antibodies and polyclonal antibodies, but the effect of the colloidal gold test strip obtained by the high manufacturing cost of the monoclonal antibodies and the polyclonal antibodies and the large batch-to-batch difference of the two antibodies is different, the nano antibodies are stable, low in production cost and can be obtained rapidly, the nano antibodies are used as markers, the analysis of small-molecule pesticide residues by combining the nano antibodies with the immunochromatography is not reported, and the nano antibodies combined with colloidal gold are not used in the detection of fenitrothion pesticide residues. Therefore, more products or methods capable of effectively detecting the fenitrothion pesticide residue are researched and developed, and the method has important theoretical research and practical application significance for developing a rapid-detection pesticide multi-residue detection technology.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the problems and provide a preparation method and application of a fenitrothion nanobody-colloidal gold marker.
The first object of the invention is to provide a preparation method of fenitrothion nanobody-colloidal gold marker.
The second object of the invention is to provide a fenitrothion nanobody-colloidal gold marker.
The third object of the invention is to provide the application of the fenitrothion nanobody-colloidal gold marker.
The fourth object of the invention is to provide a nano antibody-colloidal gold test strip for detecting fenitrothion pesticide.
A fifth object of the invention is to provide the use of the test strip.
The above object of the present invention is achieved by the following technical scheme:
according to the invention, a colloidal gold solution which can be stably combined with the fenitrothion nano-antibody is screened out through research, and the fenitrothion nano-antibody is combined with colloidal gold through optimized screening, so that the fenitrothion nano-antibody-colloidal gold marker is prepared, and has high sensitivity and strong stability.
The invention provides a preparation method of a fenitrothion nanobody-colloidal gold marker, which comprises the steps of adding the fenitrothion nanobody into a colloidal gold solution, uniformly mixing, standing, sealing, standing, centrifuging, discarding supernatant, and re-suspending precipitation by using a colloidal gold compound solution to obtain the fenitrothion nanobody-colloidal gold marker, wherein the colloidal gold solution is prepared by 1% chloroauric acid and 1% trisodium citrate in a volume ratio of 1:2, the consumption of the fenitrothion nanobody is 5-10 mug/mL, sealing is carried out for 25-40 min by adopting 25-35 mu L of 10% bovine serum albumin, and the formula of the colloidal gold compound solution is 0.02-0.5M BB, 0.1-2% BSA, 1-15% sucrose, 0.1-2% triton-100 and 0.1-2% PVP.
Preferably, the fenitrothion-resistant nanobody is prepared by referring to the prior art (Wang Yu, zhang Yifeng, shen Yudong, etc. the preparation of the fenitrothion-resistant biotinylated nanobody and the application thereof in an immunoassay method [ J ]. Modern food science and technology, 2021, volume 37 (8): 286-294, 285.).
Preferably, the pH of the colloidal gold solution is 6.5-7.5.
Preferably, the formula of the colloidal gold complex solution comprises 0.2M BB, 0.5% -2% of BSA, 2% -10% of sucrose, 0.5% -2% of triton-100 and 0.2% -1% of PVP.
Preferably, the precipitate is resuspended in 1/10 of the colloidal gold volume of the colloidal gold complex solution.
Preferably, the centrifugation conditions are 4 ℃, 8000-10000 rpm and the centrifugation time is 10-20 min.
The invention provides the fenitrothion nano antibody-colloidal gold marker prepared by the method.
The invention provides application of the fenitrothion nano antibody-colloidal gold marker in detecting fenitrothion pesticides.
The invention provides application of the fenitrothion nano antibody-colloidal gold marker in preparing a nano antibody-colloidal gold test strip for detecting fenitrothion.
The invention provides a nanobody-colloidal gold test strip for detecting fenitrothion pesticide, which contains the fenitrothion nanobody-colloidal gold marker.
The invention provides a nano antibody colloidal gold test strip for detecting fenitrothion pesticide, which comprises a sample absorption pad, a combination release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line coated with a hapten-carrier protein conjugate of the fenitrothion pesticide and a quality control line coated with a rabbit anti-camel anti-antibody, and the combination release pad is sprayed with a fenitrothion nano antibody-colloidal gold marker.
The test strip is prepared by the following steps:
s1, preparing a fenitrothion nano antibody-colloidal gold marker by adopting the method;
S2, soaking the combined release pad in a colloidal gold complex solution, drying, spraying the fenitrothion nano antibody-colloidal gold marker prepared in the step S1 on the treated combined release pad, drying, sealing, drying and preserving;
s3, coating the fenitrothion pesticide hapten-lactoferrin conjugate on a reaction membrane to form a detection line, and coating the rabbit anti-camel antibody on the reaction membrane to form a quality control line;
s4, soaking the sample absorption pad in buffer solution, and drying for later use;
s5, sequentially assembling the sample absorption pad, the combination release pad, the reaction membrane and the water absorption pad on the bottom plate to obtain the nano antibody colloidal gold test strip for detecting the fenitrothion pesticide.
Preferably, the adding amount of the fenitrothion nano antibody in the step S1 is 3-10 mug/mL.
More preferably, the adding amount of the fenitrothion nanobody is 5-10 mug/mL.
Preferably, in the step S1, a blocking reaction is performed for 25-40 min by adopting 20-40 mu L of 10% bovine serum albumin.
More preferably, 25-35 mu L of bovine serum albumin is adopted for blocking reaction for 30min.
Preferably, the concentration of the fenitrothion pesticide hapten-lactoferrin conjugate adopted in the step S2 is 0.1-0.5 mg/mL, the coating amount is 0.5-1.0 mu L/cm, the concentration of the rabbit anti-camel antibody is 0.5-2 mg/mL, and the coating amount is 0.5-1.0 mu L/cm.
More preferably, the concentration of the fenitrothion pesticide hapten-lactoferrin conjugate is 0.25mg/mL, the coating amount is 0.8 mu L/cm, the concentration of the rabbit anti-camel antibody is 1mg/mL, and the coating amount is 0.8 mu L/cm.
Preferably, the buffer in step S4 is a phosphate buffer containing 0.2% sodium chloride, 0.5% sucrose, 0.3% polyvinylpyrrolidone and 0.5% tween-20 at a ph of 7.4.
Preferably, the fenitrothion pesticide hapten-carrier protein is obtained by coupling a fenitrothion pesticide hapten and lactoferrin.
More preferably, the fenitrothion pesticide hapten solution is prepared by a description method of Chen et al analysis, 2021,146 (3): 864-873. The fenitrothion antibody solution is prepared by a description in reference to a conventional method (Wang Yu et al, modern food science, 2021,37 (8): 286-294).
Preferably, the rabbit anti-camel antibody is obtained by immunizing rabbits with nanobodies.
Preferably, the bottom plate adopts a PVC plate which does not absorb water, one side of the plastic plate is coated with self-adhesive, and the bottom plate plays a role in fixing other components of the supporting test paper.
Preferably, the sample absorbing pad may be a suction filter paper or a oil filter paper.
Preferably, the absorbent pad is preferably glass fiber paper, filter paper or absorbent paper.
Preferably, the reaction membrane may be a nitrocellulose membrane or a cellulose acetate membrane.
The invention provides a nano antibody colloidal gold test strip for detecting fenitrothion pesticide, which is characterized in that a sample solution is added into a test strip from a sample absorption pad by utilizing a high-specificity reaction and competition inhibition immunochromatography technology of fenitrothion nano antibody and fenitrothion pesticide, the sample solution flows through the whole test strip by capillary force, a substance to be detected in the sample is combined with a fenitrothion nano antibody-colloidal gold marker on a combined release pad to form a drug-antibody-colloidal gold marker, then a mixed solution flows through a reaction membrane, and the drug competes with a fenitrothion pesticide hapten-carrier protein on a detection line to combine with the fenitrothion nano antibody-colloidal gold marker, so that the residue of the fenitrothion pesticide in the sample solution to be detected is judged according to whether the color development or the color development depth of the detection line.
The invention provides application of the nanobody-colloidal gold test strip for detecting fenitrothion pesticide in detecting fenitrothion pesticide residue.
The invention has the following beneficial effects:
The invention provides a preparation method of a fenitrothion nano antibody-colloidal gold marker, and the fenitrothion nano antibody-colloidal gold marker prepared by the method has high sensitivity and strong stability. The nano antibody-colloidal gold test strip for detecting fenitrothion pesticide provided by the invention has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for rapid detection in the market, easiness in storage and long shelf life. The nanometer antibody-colloidal gold test strip for detecting the fenitrothion pesticide, which is prepared by the method, has the sensitivity reaching 0.013-0.309 mg/kg in the detection of the fenitrothion pesticide residue, the reaction time being 5-8 min, and the method for detecting the fenitrothion pesticide residue is simple, convenient, quick, visual and accurate, wide in application range, low in cost, easy to popularize and use, and is suitable for screening and on-site monitoring of a large number of samples on the market.
Drawings
FIG. 1 shows five colloidal gold solutions prepared in different volume ratios;
FIG. 2 shows the results of binding five colloidal gold solutions with different volume ratios to fenitrothion nanobody;
FIG. 3 shows the results of testing the fenitrothion drug solution by preparing colloidal gold-fenitrothion nanobody probes from five different colloidal gold solutions;
FIG. 4 is a pH optimization for the preparation of fenitrothion nanobody-colloidal gold labels;
FIG. 5 shows the optimization of the addition amount of the nanobody for preparing the fenitrothion nanobody-colloidal gold label;
FIG. 6 shows the optimization of BSA content of fenitrothion nanobody-colloidal gold labels;
FIG. 7 is a schematic illustration of the preparation of a cartap nanobody-colloidal gold conjugate optimized for colloidal gold reconstitution buffers;
FIG. 8 is a schematic diagram of a test strip;
FIG. 9 is a schematic diagram of test strip detection results;
FIG. 10 shows the results of test strips for different organophosphorus pesticides;
FIG. 11 is a standard curve of fenitrothion pesticides at different concentrations;
FIG. 12 shows the detection results of fenitrothion standard pesticides with different concentrations;
FIG. 13 is a test strip stability test result;
FIG. 14 shows test strips for different samples.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 preparation and optimization of fenitrothion nanobody-colloidal gold marker
1. Preparation of the solution
0.02-0.5M BB, 0.1-2% BSA, 1-15% sucrose, 0.1-2% triton-100 and 0.1-2% PVP.
The fenitrothion-resistant nano antibody is prepared by referring to the prior art (Wang Yu, zhang Yifeng, shen Yudong, etc. the preparation of the fenitrothion-resistant biotinylated nano antibody and the application of the fenitrothion-resistant nano antibody in an immunodetection method [ J ]. Modern food science and technology, 2021, volume 37 (8): 286-294, 285.).
2. Preparation and optimization of colloidal gold
In the prior art, monoclonal antibodies are often detected by preparing a colloidal gold solution from 1% chloroauric acid and 1% trisodium citrate in a volume ratio of 1:1, 1:1.25 and 1:1.5. Because the invention adopts the fenitrothion nano antibody, in order to prepare the colloidal gold solution suitable for the fenitrothion nano antibody, 1 percent chloroauric acid and 1 percent trisodium citrate with different volume ratios are adopted to prepare the colloidal gold solution. The particle size of the colloidal gold solution finally obtained in the preparation process of the colloidal gold solution can be changed along with the content of the trisodium citrate. Meanwhile, the volume ratio of 1% chloroauric acid to 1% trisodium citrate is 1:1, 1:1.25, 1:1.5 and 1:2 and 1:6, five colloidal gold solutions with different particle sizes are prepared and are respectively combined with the fenitrothion nanobody marks, and the combination condition of the colloidal gold solutions and the fenitrothion nanobody is judged according to the result.
Specifically, gold chloride with the mass fraction of 1% and trisodium citrate with the mass fraction of 1% are added into ultrapure water to prepare a colloidal gold solution, the colloidal gold solutions with different particle sizes can be obtained by changing the proportion of the gold chloride to the trisodium citrate, and five different volume ratios of 1:1, 1:1.25, 1:1.5, 1:2 and 1:6 are adopted to prepare the colloidal gold solutions with different particle sizes. Firstly, adding 1mL of 1% chloroauric acid into 100mL of ultrapure water, heating and stirring to boil by adopting a magnetic stirrer, then adding 1, 1.25, 1.5, 2 and 6mL of 1% trisodium citrate respectively, continuing stirring at a constant speed, changing the color of the solution from pale yellow to colorless, gradually changing into reddish wine after changing into black, stopping heating after reacting for 10min, cooling to room temperature, recovering to 100mL by using ultrapure water, and storing at 4 ℃. The prepared five colloidal gold solutions with different volume ratios are shown in figure 1, and the prepared colloidal gold has pure appearance, is transparent and has no sediment or floating matters.
The combination results of five colloidal gold solutions with different volume ratios and the fenitrothion nanobody are shown in figure 2, after the conventional colloidal gold solution with the volume ratios (1:1, 1:1.25 and 1:1.5) is adopted, the color of the colloidal gold solution is changed from red to purple and black, which indicates that the mixed solution of the colloidal gold and the nanobody is not stable enough, after the nanobody is added into the colloidal gold solution with the volume ratios of 1:2 and 1:6, the colloidal gold-fenitrothion nanobody probe is prepared from the five colloidal gold solutions with different volume ratios, and the detection results of the fenitrothion drug solution are shown in figure 3, so that the following examples all select the colloidal gold solution with the volume ratio of 1% chloroauric acid and 1% trisodium citrate for preparation and use in the subsequent steps.
3. Preparation and optimization of fenitrothion nano antibody-colloidal gold marker
The prepared colloidal gold solution is adopted, the pH value of the colloidal gold solution is regulated by potassium carbonate solution, cartap nano-antibody is added into the colloidal gold solution with regulated pH value for standing reaction for 30min, 10% BSA is added for sealing reaction for 30min, the supernatant is centrifugally discarded after sealing is finished, and the precipitate is resuspended by colloidal gold complex solution with the volume of 1/10 of that of the colloidal gold and is stored at 4 ℃.
The preparation conditions of the fenitrothion nanobody-colloidal gold marker are optimized, and the optimized result is determined according to the sensitivity and stability of the fenitrothion nanobody-colloidal gold marker finally obtained. The specific optimization conditions are as follows:
(1) pH optimization, namely, adjusting the pH of the colloidal gold solution by using 0.2M potassium carbonate solution, and adding 5, 10, 15, 20 and 25 mu L of potassium carbonate solution to obtain the colloidal gold solution with different pH values. And then obtaining different colloidal gold complexes according to the reaction process, and determining proper pH according to the sensitivity and stability of the finally obtained fenitrothion nanometer antibody-colloidal gold marker.
The pH optimization result is shown in fig. 4, when 5-15 mu L of potassium carbonate is adopted, the pH is 6.5-7.5, and the finally obtained fenitrothion nano antibody-colloidal gold marker effect can reach the detection requirement.
(2) The addition amount of the nano antibody is optimized, 3, 5,7 and 10 mu L of 1mg/mL of fenitrothion nano antibody solution is added according to the optimized pH value to obtain different colloidal gold complexes, and the proper addition amount of the fenitrothion nano antibody is determined according to the sensitivity and stability of the finally obtained fenitrothion nano antibody-colloidal gold marker.
The optimized result of the nano antibody addition amount is shown in fig. 5, and when 5-10 mu L is adopted, the effect of the finally obtained fenitrothion nano antibody-colloidal gold marker can reach the detection requirement.
(3) BSA content optimization, namely, blocking is carried out by using 20, 25, 30, 35 and 40 mu L10% BSA in a blocking process according to the optimized conditions, and the proper BSA addition amount is determined according to the sensitivity and stability of the finally obtained fenitrothion nanobody-colloidal gold marker.
As shown in FIG. 6, when 25-35 mu L of 10% BSA is adopted, the effect of the fenitrothion nanobody-colloidal gold marker finally obtained can meet the detection requirement.
(4) And optimizing the centrifugal condition according to the optimized condition, wherein the centrifugal condition comprises centrifugal temperature, centrifugal rotating speed and centrifugal time. The temperature comprises 0, 4, 16, 25 and 37 ℃, the rotating speed comprises 8000, 9000, 10000 and 12000rpm, and the centrifugation time comprises 10, 15 and 20min. And determining proper centrifugal conditions according to the sensitivity and stability of the finally obtained fenitrothion nanobody-colloidal gold marker.
And when the centrifugal condition is 4 ℃, 8000-10000 rpm and the centrifugal time is 10-20 min, the detection effect of the fenitrothion nanobody-colloidal gold marker finally obtained meets the requirements.
(5) The formula of the colloidal gold complex solution is optimized, namely the colloidal gold complex solution is optimized according to the optimal conditions, and the optimization comprises the selection of a system, the concentration of BB, the BSA content, the sucrose content, the triton-100 content and the PVP content. The system comprises BB, PB, tris, EDTA solution, concentration of the system comprises 0.02, 0.05, 0.1, 0.2 and 0.5M, adding amount of sucrose comprises 1%, 2%, 5%, 10% and 15%, and content of the rest additives comprises 0.1%, 0.2%, 0.5%, 1% and 2%. The sensitivity and stability of the fenitrothion nanobody-colloidal gold marker are also utilized to determine a proper compound solution formula.
As shown in FIG. 7, when 0.2M BB solution is adopted, the adding amount of sucrose is 2-10%, the adding amount of BSA is 0.5-2%, the adding amount of PVP is 0.2-1% and the adding amount of triathlon-100 is 0.5-2%, the effect of the finally obtained fenitrothion nanobody-colloidal gold marker can reach the detection requirement.
During the optimization process, the colloidal gold solution can aggregate under unsuitable conditions, and the color changes from wine red to black and precipitates. And finally, according to the optimization result, determining that the optimal preparation condition of the cartap nanobody-colloidal gold marker prepared by the invention is that the pH is 6.5-7.5, the adding amount of the cartap nanobody is 5-10 mug/mL, the adding amount of 10% BSA is 25-35 mug, the centrifugation condition is 4 ℃, 8000-10000 rpm and the centrifugation time is 10-20 min. The colloidal gold complex solution formula adopts 0.2M BB, 0.5-2% of BSA, 2-10% of sucrose, 0.5-2% of triton-100:0.5-2% of PVP and 0.2-1% of PVP, and the effect of the finally obtained fenitrothion nano antibody-colloidal gold marker can reach the detection requirement.
Example 2 preparation of nanobody-colloidal gold test strip for detecting fenitrothion pesticide
1. Preparation of bond release pad
Soaking the bonding release pad in a colloidal gold complex solution for 30min, placing in a 45 ℃ oven for drying, spraying the prepared fenitrothion nanobody-colloidal gold marker solution on the bonding release pad treated by the colloidal gold complex solution by adopting a AUTOKUN film-drawing metal-spraying machine (HGS 510, hangzhou peak-to-average technology Co., ltd.) at the amount of 5 mu L/cm per hole, placing the sprayed bonding release pad in the oven, drying at 37 ℃ for 12h, sealing the sprayed bonding release pad, and adding a drying agent for storage.
2. Preparation of reaction film
The preparation method of the fenitrothion pesticide hapten adopted by the invention refers to the prior art for preparation (Chen et al analysis, 2021,146 (3): 864-873.).
The fenitrothion pesticide hapten-lactoferrin is obtained by adopting chemical reagent DMF and NHS to be chemically coupled, and the rabbit anti-camel antibody is obtained by immunizing rabbits with fenitrothion nanometer antibodies.
(1) Coating a detection line, namely coating the prepared fenitrothion pesticide hapten-lactoferrin conjugate on a reaction film to form the detection line, diluting the fenitrothion pesticide hapten-lactoferrin conjugate to 0.25mg/mL by using a phosphoric acid buffer solution, coating the fenitrothion pesticide hapten-lactoferrin conjugate on a detection line (T line) on a nitrocellulose film by using a AUTOKUN film-drawing metal-spraying machine, wherein the coating amount is 0.8 mu L/cm, and drying the coated reaction film for 4 hours at 37 ℃ for standby.
(2) Coating a quality control line, namely coating the rabbit anti-camel antibody on a reaction film to form the quality control line, diluting the rabbit anti-camel antibody to 1mg/mL by using a phosphate buffer solution, coating the rabbit anti-camel antibody on the quality control line (C line) on a nitrocellulose film by using a AUTOKUN film-drawing metal-spraying machine, wherein the coating amount is 0.8 mu L/cm. And (5) drying the coated reaction film for 4 hours at 37 ℃ for later use.
3. Preparation of sample absorbent pad
The sample absorbent pad was placed in a phosphate buffer of pH7.4, 0.2mol/L containing 0.2% sodium chloride, 0.5% sucrose, 0.3% polyvinylpyrrolidone and 0.5% Tween-20, soaked for 1h and baked at 37℃for 4h for further use.
4. Assembly of test strips
The prepared reaction film, the combined release pad, the sample absorption pad and the water absorption pad are sequentially stuck on the PVC bottom plate, the initial end of the sample absorption pad is aligned with the initial end of the PVC bottom plate, the tail end of the sample absorption pad is connected with the initial end of the combined release pad, the tail end of the combined release pad is connected with the initial end of the reaction film, the sample absorption pad and the combined release pad are overlapped by 2mm, the tail end of the reaction film is connected with the initial end of the water absorption pad, the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate, the combined release pad and the water absorption pad are overlapped by 2mm, and the assembled test paper strip is shown in figure 8. And a detection line (T line) and a quality control line (C line) are arranged on the reaction film, the detection line and the quality control line are strip-shaped strips perpendicular to the length of the test strip, the detection line is positioned at one side close to the tail end of the sample absorption pad, and the quality control line is positioned at one side close to the head end of the water absorption pad. After the test paper strip is assembled according to the steps, all parts are assembled according to compaction, cut into small strips with the width of 4mm by a machine, and are arranged in a special sealing bag, and can be stored for 12 months at the temperature of 4-30 ℃.
Example 3 Limit test of nanobody-colloidal gold test strip for detecting fenitrothion pesticide
1. Specificity test
In order to detect the specificity of the nano antibody-colloidal gold test strip for detecting fenitrothion pesticide prepared by the invention, different pesticides are respectively adopted for detection. 6.5mg/kg of quetiapine, triazophos, amifos, profenofos, methomyl, carbofuran and other medicines are respectively adopted for detection, and the medicines are diluted to 6.5mg/kg by PBS solution.
And (3) vertically adding a sample solution from a sample pad of the test strip, starting timing when the liquid flows, reacting for 5-8 min, reading a result by a colloidal gold analyzer, and when the concentration of a substance to be tested in the sample is lower than a detection limit, the result is negative, and when the concentration of the substance to be tested in the sample is equal to or higher than the detection limit, the result is positive, and if the result is invalid, the retest is required.
The flow reaction principle of the test strip is shown in fig. 9, when the detection result is negative, the colors of the detection line (T line) and the quality control line (C line) are changed and become red and dark, when the detection result is positive, the detection line (T line) is unchanged or becomes light, the color of the quality control line (C line) is changed and becomes red and dark, and when the colors of the detection line (T line) and the quality control line (C line) are unchanged or only the detection line (T line) is changed and becomes red and dark, the test strip is invalid, and the test strip is required to be retested.
The test strip prepared by the invention has different pesticides, the test result is shown in figure 10, the color of the quality control line is deepened when the test strip is used for detecting fenitrothion pesticides, the result is positive, and the quality control line and the detection line are both developed and negative when other organophosphorus pesticides are detected. The test strip has no cross reaction to 6.5mg/kg of quetiapine, triazophos, fenpyrad, profenofos, methomyl, carbofuran and other medicines, and can only specifically detect fenitrothion pesticide.
2. Detection limit test
In order to detect the detection limit and sensitivity of the nano antibody-colloidal gold test strip for detecting the fenitrothion pesticide, which is prepared by the invention, fenitrothion standard pesticides are respectively added into PBS buffer solution until the final concentration is 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0156, 0.0078 and 0.0039mg/kg, and then the test strip prepared in the example 1 is taken for detection, and each sample is repeatedly tested for three times. And vertically dripping the sample solution onto the sample absorption pad, starting timing when the liquid flows, reacting for 5-8 min, and judging the result.
Accurate data can be obtained from the detection result through the reading of the colloidal gold analyzer. A standard curve can be obtained by Origin software from this data as shown in FIG. 11. The detection results of the fenitrothion standard pesticides with different concentrations are shown in figure 12, and the results corresponding to the standard curves show that the detection range of the fenitrothion pesticide residue detected by the method is 0.013-0.309 mg/kg, and the detection limit is 0.00598mg/kg, so that the national standard detection requirement is met.
3. Stability test
In order to detect the stability of the nano antibody-colloidal gold test strip for detecting the fenitrothion pesticide, which is prepared by the invention, the accuracy and the stability of the test strip are detected after the test strip is placed for a long time.
The test strip manufactured by the method is put into a sealing bag, a drying agent is arranged in the sealing bag, the sealing bag is stored in a baking oven at 45 ℃, and the sealing bag is taken out after 3 days, 7 days, 14 days, 21 days and 30 days respectively, so that the stability of the test strip is detected.
As shown in FIG. 13, the test results were not significantly changed when the test results were stored at 45℃for 3 days, 7 days, 14 days, 21 days, or 30 days, and the test results were stored at 45℃for 30 days, which corresponds to 12 months at normal temperature.
Example 4 actual sample detection
Different cabbage, cucumber and orange samples are purchased from markets and supermarkets, the samples are cleaned and dried, then 10g (+ -0.1 g) of homogeneous samples are weighed into a 50mL plastic centrifuge tube, the samples are divided into negative samples and positive samples, no additional treatment is carried out on the negative samples, and the cartap standard is added into the positive samples until the drug concentration reaches 0.02mg/kg. Then adding 10mL of acetonitrile into the negative sample and the positive sample, mixing uniformly by vortex, then adding an extraction bag, continuing vortex shaking for 2min, centrifuging for 3min at 5000rpm/min, and standing at room temperature. Adding the supernatant solution into a purifying tube, vortex shaking for 2min, centrifuging for 3min at 5000rpm/min, collecting supernatant, blowing with 37 deg.C nitrogen, and dissolving again with EDTA solution.
And then taking the test strip prepared in the embodiment 2 for detection, vertically dripping the treated sample solution onto the sample absorption pad, starting timing when the liquid flows, reacting for 5-8 min, and judging the result.
The detection result is read by a colloidal gold analyzer:
Negative (-) indicates that the concentration of the substance to be detected in the sample is lower than the detection limit;
positive (+)'s means that the concentration of the substance to be detected in the sample is equal to or higher than the detection limit;
Invalidation-indicates that retest is required.
When the test strip prepared by the invention is used for detecting different samples, the detection result is shown in fig. 14, when detecting the Chinese cabbage, the cucumber and the orange samples, the T line and the C line in the negative sample can be seen to develop, and the C line of the positive sample is obviously stronger than the development result of the T line, so that the detection result can meet the requirements.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (8)
1. A cartap nanobody-colloidal gold marker is prepared by adding cartap nanobody into a colloidal gold solution, uniformly mixing, standing, sealing, standing, centrifuging, discarding supernatant, and re-suspending sediment by using a colloidal gold compound solution to obtain the cartap nanobody-colloidal gold marker, wherein the colloidal gold solution is prepared by 1% chloroauric acid and 1% trisodium citrate in a volume ratio of 1:2, the pH of the colloidal gold solution is 6.5-7.5, the adding amount of the cartap nanobody is 5-10 mug/mL, sealing reaction is carried out for 25-40 min by adopting 10% bovine serum albumin 25-35 mug, and the formula of the colloidal gold compound solution is 0.02-0.5 MBB, 0.1-2% BSA, 1-15% sucrose, 0.1-2% triton and 0.1-2% PVP.
2. The fenitrothion nanobody-colloidal gold marker according to claim 1, wherein the formula of the colloidal gold complex solution comprises 0.2M BB, 0.5% -2% BSA, 2% -10% sucrose, 0.5% -2% triamcinolone acetonide-100 and 0.2% -1% PVP.
3. The use of the fenitrothion nanobody-colloidal gold marker according to claim 1 in the detection of fenitrothion pesticides.
4. The use of the fenitrothion nanobody-colloidal gold label according to claim 1 in preparing a nanobody-colloidal gold test strip for detecting fenitrothion.
5. A nanobody-colloidal gold test strip for detecting fenitrothion pesticide is characterized by comprising the fenitrothion nanobody-colloidal gold marker as set forth in claim 1.
6. The test strip of claim 5, wherein the test strip is prepared by the steps of:
s1, preparing a fenitrothion nanobody-colloidal gold marker by adopting the method of claim 1;
S2, soaking the combined release pad in a colloidal gold complex solution, drying, spraying the fenitrothion nano antibody-colloidal gold marker prepared in the step S1 on the treated combined release pad, drying, sealing, drying and preserving;
s3, coating the fenitrothion pesticide hapten-lactoferrin conjugate on a reaction membrane to form a detection line, and coating the rabbit anti-camel antibody on the reaction membrane to form a quality control line;
s4, soaking the sample absorption pad in buffer solution, and drying for later use;
s5, sequentially assembling the sample absorption pad, the combination release pad, the reaction membrane and the water absorption pad on the bottom plate to obtain the nano antibody colloidal gold test strip for detecting the fenitrothion pesticide.
7. The test strip of claim 6, wherein the concentration of the fenitrothion pesticide hapten-lactoferrin conjugate adopted in the step S2 is 0.1-0.5 mg/mL, the coating amount is 0.5-1.0 mu L/cm, the concentration of the rabbit anti-camel antibody is 0.5-2 mg/mL, and the coating amount is 0.5-1.0 mu L/cm.
8. The use of the nanobody-colloidal gold test strip for detecting fenitrothion pesticide according to claim 5 in detecting fenitrothion pesticide residue.
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| CN103018435A (en) * | 2012-07-11 | 2013-04-03 | 南京农业大学 | Organophosphorus pesticide multiresidue fast immune-gold localization test strip and preparation method thereof |
| CN112505321A (en) * | 2020-10-23 | 2021-03-16 | 重庆中元汇吉生物技术有限公司 | Redissolution of antibody-labeled carrier |
| CN113388037A (en) * | 2021-04-14 | 2021-09-14 | 华南农业大学 | Preparation and application of specific recognition fenitrothion nano antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103018435A (en) * | 2012-07-11 | 2013-04-03 | 南京农业大学 | Organophosphorus pesticide multiresidue fast immune-gold localization test strip and preparation method thereof |
| CN112505321A (en) * | 2020-10-23 | 2021-03-16 | 重庆中元汇吉生物技术有限公司 | Redissolution of antibody-labeled carrier |
| CN113388037A (en) * | 2021-04-14 | 2021-09-14 | 华南农业大学 | Preparation and application of specific recognition fenitrothion nano antibody |
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